The overall objective of this project is to examine the pathways that mediate mu-opioid receptor regulation and opioid tolerance in the intact animal. It has been established that opioid agonists differ in their ability to regulate CNS mu-opioid receptors in vivo. Chronic treatment with high intrinsic efficacy opioid agonists (e.g., etorphine) downregulates mu opioid receptors, while at the same time producing tolerance and regulation of mu-opioid receptor mRNA levels. Chronic treatment with lower intrinsic efficacy opioid agonists (e.g., morphine) can induce tolerance without altering mu-receptor density or mu-receptor mRNA. At present, the basis for the different effects of treatment with high and low intrinsic efficacy opioid agonists in vivo are not known. However, compelling data raise the possibility that opioid tolerance and mu-opioid receptor regulation require the activation of different intracellular signaling systems. This project will test the hypothesis that opioid agonist-induced downregulation of mu-opioid receptors and regulation of mu-opioid receptor mRNA levels in vivo involves three signaling proteins: G-protein receptor kinase 2,beta arrestin 2 and dynamin. It is further hypothesized that morphine-induced tolerance is independent of this signaling pathway in vivo. Finally, it is hypothesized that the magnitude of opioid tolerance is increased by mu-opioid receptor downregulation. The experiments proposed in this application will use behavioral, biochemical and molecular pharmacological methods to determine the role of these proteins in mediating opioid agonist-induced tolerance, downregulation of mu-opioid receptors and regulation of mu- receptor mRNA in vivo. The results of these studies will significantly enhance our knowledge of the pathways that regulate chronic opioid effects in the intact, behaving animal. As such, these results may provide important insights for developing strategies to treat opioid drug abuse and pain.